Inhibitor of rna polymerase ii

ABSTRACT

An inhibitor of RNA polymerase II is described, wherein said inhibitor is selected a moiety which targets a protein selected from cyclin kinase 12 (CDK12) or its recruiting protein PAF1C. Particular examples of such inhibitors are polypeptides expressed by a gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, such as a commensal bacteria or asymptomatic carrier, or a variant of said protein. Inhibitors may be based upon bacterial Sigma S or NplD proteins. These inhibitors are useful in therapies, to suppress protein expression. Thus they may be used as immunosuppressants, anti-inflammatory or anti-infection agents.

The present invention relates to factors and moieties that can modulatehost protein expression, for example, a factor having immunosuppressantactivity, to methods of preparing the factors and moieties and to theiruse in therapy.

BACKGROUND TO THE INVENTION

The multi-subunit RNA polymerase II complex (Pol II) is essential forprotein expression in eukaryotes. Transcription cycle has beenextensively characterized to show that around 10% of all expressed genesare involved in transcriptional regulation, assembly and control of thePol II complex. In eukaryotic cells, RNA polymerase II catalyzes thesynthesis of mRNA and small nuclear RNA. Pol II transcription cycleconsists of several stages, termed preinitiation, initiation, promoterclearance, during which Pol II pauses at the promoter proximal site,followed by escape from pausing, productive elongation and termination.

For specificity, the assembly of Pol II is tightly controlled and theefficiency of transcription is modified at individual promoters byactivators and repressors, as well as general or specific transcriptionfactors. The carboxy-terminal domain (CTD) of the largest Pol IIsubunit, Rpb1, contains a heptamer sequenceTyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 repeated 52 times in mammalian cells.During the transcription cycle, CTD is subject to continuous structuralremodeling by kinases and phosphatases and serves as a platform forbinding and release of numerous regulatory proteins. The recruitment ofSer2-specific kinase activity in the form of positive transcriptionelongation factor b (P-TEFb) is regarded to be a critical step in theactivation of promoter-proximally paused Pol II, facilitating itsrelease from pause sites. P-IEFb is composed of cyclin-dependent kinase9 (CDK9) and its regulatory cyclin T1. The more recently discoveredCdk12 phosphorylates CTD of RNA polymerase in the middle and 3′ end ofgenes. P-TEFb is a general transcription factor required for efficientexpression of most cellular genes, therefore, its activity is accuratelymediated with positive regulator bromodomain protein Brd4, and negativeregulators noncoding 7SK snRNA and the HEXIM1 protein. It was shown thatP-TEFb recruits PAF1 to Pol II complex, which is followed by CDK12recruitment by PAF1 (Yu et al., Science 2015. 350(6266:1383-6).De-phosphorylation of Ser2-phosphates is done by phosphatases FCP1(TFIIF-dependent CTD phosphatase 1) and Cdc14.

The host Pol II transcription machinery is targeted by bacteria, asfirst described for Escherichia coli strains that establish anasymptomatic carrier state in the human urinary bladder (Lutay et al.,J. Clin Invest. 2013, 123(6) 2366-79). Asymptomatic bacteriuria activelymodifies the host response by inhibiting Pol II dependent transcription,including pathology-generating signaling pathways in the host (Lutay etal., 2013 supra.). In 24 hours after human inoculation with theprototype ABU E. coli strain 83972, more than 60% of all genes weresuppressed; this inhibition was verified by infection of human cells.Among different ABU strains (n=75), 37% were strongly inhibitorycompared to 17% of APN strains (n=88). The symbiotic relationshipbetween ABU strains and their hosts is also influenced by a lack ofvirulence factors, resulting from virulence gene attenuation in thesestrains. These findings indicate that suppression or inhibition of RNApolymerase II may be an effective method for modulating the immunesystem, in particular by acting as modulators of gene expression. Thereis frequently a need to modulate gene expression, in particular of theimmune system either by stimulation or suppression in connection with awide variety of therapeutic applications.

Immunosuppressants are required in a wide a variety of therapies. Thisincludes for instance, the prevention of rejection of transplantedorgans or tissues such as bone marrow, heart, kidney or liver, thetreatment of autoimmune diseases such as rheumatoid arthritis, multiplesclerosis, myasthenia gravis, lupus, sarcoidosis, Crohn's disease,pemphigus and ulcerative colitis.

However, as a broad spectrum suppressor of protein expression, Pol-IIinhibitors may find application in any therapy where host protein isover-expressed or problematic. Thus inhibitors may also be used in thetreatment of inflammatory disease such as asthma, or in the treatment ofauto-inflammatory disease such as Behcet's disease, FMF, NOMID, TRAPS orDIRA. It has also been suggested that host-directed immunomodulatorytherapies can be used in the treatment of infections, whereby naturalmechanisms in the host are exploited to enhance therapeutic benefit. Inthis case, the objective is to initiate or enhance protectiveantimicrobial immunity while limiting inflammation-induced tissueinjury. Such a mechanism may help to address the increase orantimicrobial resistance as bacteria become resistant to conventionalantimicrobial drugs over time.

Earlier attempts to characterize the mechanism of Pol II inhibition bythe ABU by comparison of whole genome sequences of E. coli 83972 and theuropathogenic strain, E. coli CFT073 have failed to identify specificfactors associated with this (I. Amibite et al. Pathogens, 2016, 5, 49;doi:10.3390), indicating that this is not a simple matter. However, as aresult of the serendipitous occurrence of attenuated Pol II inhibitoryactivity in a re-isolate, obtained from a patient inoculated with aprototype ABU, the applicants have been able to make a significantbreakthrough in identification of the factors which may be useful inimmunosuppression.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided aninhibitor of host RNA polymerase II (pol II inhibitor) for use intherapy, wherein said inhibitor is a moiety which targets a proteinselected from PAF1C or CDK12.

Moieties which target these proteins may be known in the art or may beidentified or designed using conventional screening methods. They mayfor example comprise antibodies or binding fragments thereof, aptamersor small molecules, which bind to CDK12 or PAF so as to inhibit RNApolymerase II in a host cell.

Specific examples of such moieties may be obtained from bacteria, suchas the commensal bacteria or asymptomatic carriers such as asymptomaticbacteriuria.

As a result of the isolation of a spontaneous, loss of function mutantof the ABU strain E. coli 83972, the applicants have identifiedbacterial proteins able to act as direct inhibitors Pol IIphosphorylation in infected hosts. Mutations were localized bycomparative genome sequencing of the E. coli 83972 WT and mutant strainand identified sequence variants were systematically introduced into theE. coli 83972 WT strain for analyses of effects on Pol IIphosphorylation.

In a particular embodiment, the inhibitor is a polypeptide expressed bya gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, ora variant of said polypeptide.

The TATA box binding protein Sigma S was shown to bind human TATA boxDNA and to competitively inhibit the binding of human TBP, thusincapacitating the Pol II pre-initiation complex. Furthermore, NlpD,which regulates Sigma S expression, stimulated the degradation of PAF1C,which recruits the kinase CDK12 to the Pol II phosphorylation complex.Both proteins entered host cells. The results identify a novel mechanismused by bacteria to regulate fundamental host cell functions, such asthe transcriptional activity at sites of infection.

In a particular embodiment, the inhibitor is a bacterial Sigma S proteinor a variant thereof, or an active fragment of either of these. Anexample of such a protein is SEQ ID NO 2 or a truncated form of SEQ IDNO 2.

(SEQ ID NO 1) MSQNTLKVEIDLNEDAEFDENGVEVFDEKALVEQEPSDNDLAEEELLSQGATQRVLDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVASRRRMIESNLRLVVKIARRYGNRGLALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKELNVYLRTARELSEIKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKALLDILADEKENGPEDTTQDDDMKQSIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGREIGLTRERVRQIQVEGLRRLREILQTQGLNIEALFRE (SEQ ID NO 2)MFRQGITGRSFILMSQNTLKVEIDLNEDAEFDENGVEVFDEKALVEEEPSDNDLAEEELLSQGATQRVLDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVASRRRMIESNLRLVVKIARRYGNRGLALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKELNVYLRTARELSEIKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKALLDILADEKENGPEDTTQDDDMKQSIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGREIGLTRERVRQIQVEGLRRLREILQTQGLNIEALFRE

As used herein, the term ‘fragment’ refers to any portion of the givenamino acid sequence which will shows Pol II inhibitory activity.Fragments may comprise more than one portion from within the full-lengthprotein, joined together. Portions will suitably comprise at least 5 andpreferably at least 10 consecutive amino acids from the basic sequence.

Suitable fragments will include deletion mutants comprising at least 10amino acids, for instance at least 20, more suitably at least 50 aminoacids in length or analogous synthetic peptides with similar structures.They include small regions from the protein or combinations of these.

In a particular embodiment, the fragment will be a peptide whichinhibits binding of TBP to Sigma S, for example a fragment comprisingamino acids 149-183 of SEQ ID NO 2, shown in bold type in the abovesequence, which forms SEQ ID NO 3.

Certain such pol II inhibitors will be novel and these form a furtheraspect of the invention.

They may be used in a range of therapeutic applications as discussedabove, to suppress protein expression. In particular, they may be usedas immunosuppressant, anti-inflammatory or anti-infection (such asantibacterial) agents.

The expression “variant” refers to proteins or polypeptides having asimilar biological function but in which the amino acid sequence differsfrom the base sequence from which it is derived in that one or moreamino acids within the sequence are substituted for other amino acids.Amino acid substitutions may be regarded as “conservative” where anamino acid is replaced with a different amino acid with broadly similarproperties. Non-conservative substitutions are where amino acids arereplaced with amino acids of a different type.

By “conservative substitution” is meant the substitution of an aminoacid by another amino acid of the same class, in which the classes aredefined as follows:

Class Amino acid examples Nonpolar: A, V, L, I, P, M, F, W Unchargedpolar: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

As is well known to those skilled in the art, altering the primarystructure of a polypeptide by a conservative substitution may notsignificantly alter the activity of that polypeptide because theside-chain of the amino acid which is inserted into the sequence may beable to form similar bonds and contacts as the side chain of the aminoacid which has been substituted out. This is so even when thesubstitution is in a region which is critical in determining thepeptide's conformation.

Non-conservative substitutions are possible provided that these do notinterrupt the function of the DNA binding domain polypeptides. Broadlyspeaking, fewer non-conservative substitutions will be possible withoutaltering the biological activity of the polypeptides. Determination ofthe effect of any substitution (and, indeed, of any amino acid deletionor insertion) is wholly within the routine capabilities of the skilledperson, who can readily determine whether a variant polypeptide retainsthe fundamental properties and activity of the basic polypeptide. Forexample, when determining whether a variant of the polypeptide fallswithin the scope of the invention, the skilled person will determinewhether complexes comprising the variant retain biological activity(e.g. tumour cell death) of complexes formed with unfolded forms of thenative protein and the polypeptide has at least 60%, preferably at least70%, more preferably at least 80%, yet more preferably 90%, 95%, 96%,97%, 98%, 99% or 100% of the native protein.

Variants of the polypeptide may comprise or consist essentially of anamino acid sequence with at least 70% identity, for example at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% identity toa native polypeptide sequence. The level of sequence identity issuitably determined using the BLASTP computer program with the nativepolypeptide sequences as the base sequence. This means that nativepolypeptide sequences form the sequence against which the percentageidentity is determined. The BLAST software is publicly available athttp://blast.ncbi.nlm.nih.gov/Blast.cgi (accessible on 13 Oct. 2016).

In a particular embodiment, the inhibitor is a polypeptide expressed bya gene selected from IldD, IldR, nlpD or rfaH of a bacterial species.The bacterial species is suitably a commensal bacteria or anasymptomatic carrier such as asymptomatic bacteriuria (ABU). Inparticular, the bacteria is an E. coli strain, such as E. coli 83972.

In a particular embodiment also, the inhibitor is a polypeptideexpressed by an IldD, Ildr or nldD gene and secreted by the bacteria.

In some embodiments, the inhibitor is of low molecular weight forinstance less than 3 kDa. It is suitably resistant to Proteinase K.

In other embodiments, the inhibitor is a larger protein, for example ofabout 40 kDa. Such inhibitors may be obtained by culturing the bacteriain an appropriate culture medium such as RPMI. RNA polymerase IIactivity, which may be tested using appropriate testing methods such asthose exemplified hereinafter, may be found in the supernatant,suggesting that the factor responsible is secreted by the bacteria.Suitably the supernatant is separated from the bacteria for exampleusing centrifugation as a preliminary step and then subjected toanalysis to confirm RNA polymerase II inhibitor activity. Filtration ofthe supernatant for example using centrifugal or flow-through filterssuitable for separating proteins can be used to remove non-activefractions which tend to be high molecular weight components, thusconcentrating the inhibitor activity.

The inhibitor may be purified from the concentrate using conventionalmethods and the amino acid sequence determined, also using conventionalmethods. When this has been done, the inhibitor may be producedsynthetically, for example using recombinant DNA technology.

For use in modulating the immune system, the pol II inhibitor of theinvention is suitably formulated into a pharmaceutical composition inwhich it is combined with a pharmaceutically acceptable carrier. Suchcompositions form a further aspect of the invention.

The composition may be in a form suitable for oral administration, forexample as a tablet or capsule, for parenteral injection (includingintravesical, intravenous, subcutaneous, intramuscular, intravascular orinfusion) as a sterile solution, suspension or emulsion, for topicaladministration as an ointment or cream or for rectal administration as asuppository.

In general the above compositions may be prepared in a conventionalmanner using conventional excipients.

Methods of using the RNA polymerase II inhibitor will depend upon avariety of factors such as the disease being treated and the nature ofthe particular inhibitor being used. These will be determined inaccordance with clinical practice.

In yet a further aspect, the invention provides a method of treating apatient in need therefore with an effective dosage of an inhibitor ofRNA polymerase II as described above. Suitable dosages will bedetermined in accordance with normal clinical practice, but willgenerally be in the range approximately from 0.01 g/kg to 25 g/kg butwith considerable variation between individuals and disease conditions.

As reported hereinafter, the mechanism of Pol II inhibition by the ABUstrain was further investigated, but using re-isolates of the prototypeABU strain from inoculated patients, which were screened for attenuationof Pol II inhibitory activity. An attenuated re isolate SN25, wassubjected to whole genome sequencing and genomic differences between theancestor and progeny were identified. The variant positions in thechromosome were subsequently mutated in E. coli 83972, by homologousrecombination and the mutants were screened for effects on Pol II Ser 2phosphorylation. The genes lldD and lldR, whose products are involved inaerobic L-lactate metabolism, were shown to significantly affect Pol IIphosphorylation. To confirm these effects, a biochemical approach wasused. Supernatant of bacteria incubated in tissue culture medium wereshown to contain inhibitory activity and was further fractionated todefine the molecules responsible for these effects. Weak acids of <3 kDmolecular weight, formic and acetic, were identified to have inhibitoryeffect of Pol II phosphorylation. In addition, Pol II phosphorylationwas affected by deletion of nlpD, which encodes a lipoprotein with apotential function in cell wall formation and rfaH and cysE withproducts of both genes involved in biofilm formation.

In particular, the applicants have identified a new mechanism by whichbacterial proteins disturb the formation of the Pol II complex andattenuate the Pol II activation in human cells. The bacterial TATA boxbinding protein, encoded by rpoS and its transcriptional regulator Nlpdas bacterial genes responsible for this effect through competitiveinhibition of the human TATA box binding protein TBP and a reduction inPAF1C, CDK 9 and CDK12. The findings define a new, potent mechanism forcross-regulation of the transcriptional machinery between eukaryotic andprokaryotic cells. Without being bound by theory, the findings mayillustrate a new general mechanism of bacterial adaptation and survivalin infected hosts.

The encounters between bacteria and host cells are guided by specificmolecular interactions. Pathogen attack is often executed by virulencefactors via receptor-mediated interactions, involving conservedreceptors like the TLRs and pathogen specific recognition mechanismswith specificity for unique sets of virulence factors. Adhesiveinteractions determine the tissue specificity and site of infection.Exotoxins bind cell surface receptors, such as GM1 (cholera toxin) orGb3 (Shiga toxin) and after internalization, the toxins interfere withkey cellular functions. Endotoxins activate TLR4 signaling cascades andby engaging specific co-receptors, virulence factors determine adaptorprotein usage and transcription factor recruitment. Here we propose thatbacteria export molecules that enter host cells and attenuate thetranscriptional machinery, by competing with molecules involved in theassembly of the Pol II complex and its phosphorylation at Ser2.

As described herein, two, closely related genes have been identified inthe WT strain E. coli 83972, based on a screen for “loss of function”mutants. A Tyr209Hi amino acid change in NlpD, abolished the inhibitoryeffect on Pol II phosphorylation. NlpD is a secreted protein with a 25aa-signal sequence and potential signal peptidase cleavage site and istransported to the bacterial periplasm. NlpD together with other LytM(lysostaphin)—domain-containing factors is required for septalproteoglycan splitting and daughter cell separation. When overexpressed,nlpD changes bacterial morphology, due to the activation of the cellwall hydrolase AmiC. The mutation in SN25 appeared to be located withinthe AmiC binding site, suggesting that this mutant has lost the abilityto activate AmiC and thus to facilitate the secretion of bacterialcomponents and their interactions with the host cell. The rapidreduction in PAF1C and CDK12 protein levels suggested that hostproteases or ubiquitinases might be activated. Uehara et al., 2010discuss four possible mechanisms of bacterial amidase activation by LytMfactors including NlpD. These include 1) allosteric or 2) covalentmodification (e.g. proteolytic processing) of amidases by the LytMfactors, 3) facilitated substrate association of the amidases and 4)prior deformation or hydrolysis of bonds in the PG substrate. Therefore,similarly to activation of the amidase in bacterial cells, NlpD mightactivate some other enzymes in the host cells.

The NlpD protein is of SEQ ID NO 4:

(SEQ ID NO 4)   1msagspkftv rriaalslvs lwlagcsdts nppapvssvn gnapantnsg mlitpppkmg  61ttstaqqpqi qpvqqpqiqa tqqpqiqpmq pvaqqpvqme ngrivynrqy gnipkgsysg 121stytvkkgdt lfyiawitgn dfrdlaqrnn iqapyalnvg qtlqvgnasg tpitggnait 181qadaaeqgvv ikpaqnstva vasqptitys essgeqsank mlpnnkptat tvtapvtvpt 241asttepivss tststpistw rwptegkvie tfgaseggnk gidiagskgq aiiatadgrv 301vyagnalrgy gnliiikhnd dylsayahnd tmlvreqqev kagqkiatmg stgtsstrlh 361feirykgksv nplrylpqr

Interestingly, NlpD is homologous to a human protein, themembrane-spanning 4-domains protein, subfamily A, member 15. A putativerole of the membrane-spanning 4-domains protein based on data-mining issignal transduction by being a component of a multimeric receptorcomplex (human gene database GeneCards). Homologous region is locatedtowards N-terminus of NlpD (amino acids 44-90) with homology andsimilarity averaging 37% and 53%, respectively. This might provide somemechanistic explanation of NlpD being potentially inserted into membraneof the host cell before reaching its targets inside the cell.

NlpD also exerts its effect through transcriptional control of rpoS andrpoS-dependent genes. The transcription of rpoS is regulated from acommon nlpD promoter and from additional sites within the nlpD ORF.Sigma S is an important regulator of more than 20 stationary phase genesand operons such as genes required for multiple-stress resistance. Inaddition, as NlpD and Sigma S proteins are encoded by the same genecluster and are derived from the same polycystronic RNA, directinteractions cannot be excluded. It can be hypothesized that NlpDallosterically modifies Sigma S, facilitating its transport andrendering it active in binding and melting of host cell DNA.

In a particular embodiment, the inhibitor comprises a bacterial NplDprotein, or a variant thereof, or an active fragment of either of these.Examples of such proteins are of SEQ ID NO 4.

Without being bound by theory, it is possible that disruption of thepre-initiation complex by Sigma S might be a key step, affectingdownstream transcriptional activity. The applicants have shown thatbacterial RpoS competes with human TBP for binding to TATA box DNA. Bydislodging TBP from its binding site, RpoS may thus preventpre-initiation complex formation and therefore the binding of Pol II tospecific promoters. A general effect on gene expression is supported bya clinical study, showing reduced expression of >60% of all genes incirculating blood cells in patients inoculated with E. coli 83972.Despite the inhibition of a large number of genes, effects were muchless pronounced than when a pharmacological inhibitor was used, however,suggesting specificity. In eukaryotic promoters, PIC formation andbinding of general transcription factor II B (TFIIB) recruits Pol II tothe promoter. II B (TFIIB) consists of 4 functional domains—N-terminalZn ribbon, B reader, B linker and Core domain. Core domain in eukaryotesis required to stabilize the TBP-DNA complex and the Zn ribbon recruitsRNA Pol II.

Amino acid analysis reveals that Sigma S shows homology to the coredomain of TFIIB (38% over the stretch of 35 AA) but no homology with theZn ribbon, potentially explaining why Sigma S binds DNA but fails torecruit eukaryotic Pol II. The findings suggest an interestingevolutionary model, where maintaining the basic organization of geneexpression and Pol II machinery constituents from bacteria to humansoffers a mechanism for coordinate regulation of gene expression betweeneucaryotes and procaryotes.

To summarize, the applicants obtained a set of data supporting thehypothesis that both bacterial proteins NlpD and Sigma S areinternalized by the human host cells. These include experiments on wholecell lysates of cells infected with ABU, co-immunoprecipitation ofPol-II, Sigma S and Nlp D, as well as immunofluorescence studies of ABUinfected kidney cells (FIGS. 6A-6D). With immunofluorescence, somebackground staining is observed for NlpD and RpoS in uninfected control,which might be explained by the relative homology between NlpD and RpoSwith some human proteins. NlpD is homologous to human protein,membrane-spanning 4-domains subfamily A member 15 (with 37% homology and53% similarity over the stretch of 47 amino acids) as well to humanprotein GREB1-like protein isoform X4, Growth Regulation By Estrogen inBreast Cancer (25% homology and 40% similarity, 116 AA). Likewise, SigmaS is homologous to two human proteins, circulating B cell antibody heavychain variable region (31% homology and 54% similarity, 45 AA) and asdiscussed earlier to transcription factor II B (38% homology and 48%similarity, 35 AA).

The invention will now be particularly described by way of example withreference to the accompanying Figures in which:

FIG. 1 illustrates inhibition of eukaryotic RNA Pol II phosphorylationby 83972 ABU strain: A. Cells were infected in suspension, labelled forPol II-Ser2 with fluorescently labeled AB and run on cell flowcytometer. The distribution of phosphorylated Pol II fluorescence wasanalyzed. Infection with the AU strain decreases host Pol IIphosphorylation; B Pol II phosphorylation in control and ABU infectedcells, visualized by laser-scanning, slides were mounted and imaged.Nuclei were counterstained with DRAQS and fluorescence intensity wasquantified by ImageJ software 1.46r. C. Mean value of Pol IIphosphorylation in ABU infected cells and uninfected control cells. Onerepresentative experiment is shown, measured with flow cytometry asshown in A and one was quantified by confocal microscopy, using ImageJsoftware 1.46r. Means of two experiments. D-E shows how a particularre-isolate, designated SN25, has lost the inhibitory effect on RNA PolII phosphorylation. Results show loss of Pol II phosphorylationrepression by reisolate strain SN25 as compared to ancestor strain 83972in A-C. Error bars show standard error of the mean. G. Hit map of geneexpression after infection with both strains shows 465 genes upregulatedand 385 genes downregulated in both ABU and SN25, 224 genesdownregulated only by ABU and 2001 genes differentially regulated bySN25, but not changed by ABU. H. Moreover, host gene expression of theinnate immune response genes was also activated more efficiently by SN25than E. coli 83972. The expression of cytokines and cytokine receptorswas enhanced in SN25-infected compared to ABU-infected human kidneycells. More highly regulated cytokines include CXCL1, 2, 3 and 8, CCLSand 10, IL1B as well as CSF2, LTB and WNTSA. The results identify SN25as a loss of Pol II inhibition mutant, with stronger pro-inflammatoryeffects than the parent strain, consistent with the loss of Pol IIinhibition.

FIG. 2 A. Schematic location of mutations in SN25 genome compared to ABU83972, B List of specific mutations found. C. List of genes mutated inSN25 and their corresponding functions.

FIG. 3 illustrates the effect on Pol II phosphorylation of ABU deletionmutant reproducing the genomic changes in SN25. The following genes weredeleted from 83972 ABU genome: lldD, lldR, nlpD, rfaH, cysE, rcsB, mdoHand lrhA. These mutants were studied with flow cytometry andfluorescence scanning microscopy for level of Pol II phosphorylation; ACells were infected in suspension, stained for Pol II-Ser2 withfluorescently labeled AB and analyzed with flow cytometry. B. Mean valueof Pol II phosphorylation in control, ABU infected sample and samplesinfected with single gene ABU mutants as measured by flow cytometry andC. by microscopy. It is shown that upon infection with some single geneABU mutants, host Pol II phosphorylation becomes higher compared to ABUinfection implying that these genes are involved in repression of hostPol II phosphorylation. Statistical difference in level of Pol IIphosphorylation was measured with T test; D-E. Experimental design aimedto identify the Pol II inhibitor; Bacteria taken from agar plates andsuspended in PBS did not show inhibitory activity; RPMI incubated with10⁹ CFU/mL of bacteria for 4 hours was collected, centrifuged to removebacterial cells and filtered through a 0.2 μm filter. The filtratecontained significant inhibitory activity (p<0.001), suggesting thatbacterial growth in cell culture medium (RPMI) induces the secretion ofinhibitor(s). Screening of supernatant of mutants revealed that allsingle gene mutants, apart from lldD, lldR, nlpD and rfaH, secrete thesubstance with inhibitory activity.

FIG. 4 illustrates how the ABU strain inhibits the Pol II Ser2 CTDphosphorylating machinery by targeting cyclin kinase 12 and itsrecruiting protein PAF1C. A. Eukaryotic phosphorylation machineryconsists of two cyclin-dependent kinases CDK9 and 12, that phosphorylateCTD of Pol II biggest subunit at Ser2 residue. PAF1C complex of proteinsrecruits CDK12 to promoter. B. Western blot of whole cell lysates afterinfection with ABU and SN25 showing decrease of expression of proteinsrequired for Pol II phosphorylation. Table inset shows % inhibition ofprotein as mean value of 2 experiments. C. Confocal microscopy of cellsinfected with ABU strain and SN25 reisolate. Cells were treated withanti-PAF1c or anti-CDK12 primary antibody and correspondingfluorescently labeled secondary antibodies. Nuclei were counterstainedwith Draq5. D. Fluorescence intensity of staining in C was quantified byImageJ software 1.46r. Mean values of two experiments are shown. E. Toidentify genes involved in PAF1c and CDK12 inhibition, cells wereinfected with single gene ABU mutants and level of PAF1C and CDK12proteins was assessed with western blot. Table below specifies % ofinhibition. NlpD was identified as gene that is primarily involved insuppression. F. Confirmation with confocal microscopy of the effect ofattenuated PAF1C and CDK12 suppression by d NlpD mutant.

FIG. 5. Sigma S as an effector molecule suppressing host geneexpression. A. NlpD gene is located upstream of rpoS, which encodesSigma S; the DNA binding subunit of bacterial RNA Polymerase. NlpDregulates Sigma S expression through internal promoter. B. An rpoSdeletion mutant was constructed in E. coli 83972. The loss of Sigma Sprotein is demonstrated by Western blot analysis of bacterial lysates ofstrains SN25, d nlpD and d rpoS. C. The rpoS deletion mutant was used toinfect human kidney cells. Confocal microscopy shows the reduction inPol II Ser2, Pafl c and CDK12 levels was attenuated compared to the ABUstrain. D. Quantification of data in C. E. Comparison of bacterial andeukaryotic type II RNA polymerase. Homologous subunits are similarlyshaded. F. A schematic illustration of the hypothesis for Sigma Sbinding to eukaryotic promoter DNA and competitive inhibition of TBPbinding. G. Testing of hypothesis in E. Human and bacterial TATA boxoligo-nucleotides were incubated with synthetic peptide covering theDNA-binding domain of Sigma S. Sigma S is shown to bind prokaryotic andhuman TATA box oligonucleotides, creating band shifts with similarmobility. Specificity was confirmed by inhibition of binding with SigmaS-specific antibodies. H. Adding of TBP with whole cell lysate to IRF3promoter DNA leads to TBP-DNA complex. Sigma S competes with TBP forbinding to IRF3 promoter as visualized by concentration dependentdecrease in intensity of shifted band.

FIG. 6. Interactions of Sigma S and NlpD with the Pol II complex A.Confocal imaging of infected human kidney cells after staining withantibodies specific for Pol II Ser2 and Sigma S. A parallel loss ofnuclear Pol II staining and accumulation of RpoS in nuclear aggregateswas detected. B. Detection by Western blots of Sigma S in whole cellextracts and the nuclear fraction of cells infected with the ABU strain.Sigma S was not detected in cells infected with the SN25 reisolate ornlpD or rpoS deletion mutants. C. Binding of Sigma S and NlpD to the PolII complex is illustrated and detected by western blot afterco-immunoprecipitation of whole cell lysates with antibodies specificfor total Pol II. D. Confocal imaging of human kidney cells, infectedwith E. coli 83972WT and stained with antibodies specific for Sigma S orPol II, with nuclear DRAQ5 counterstaining.

FIG. 7. Functional relevance confirmed with in vivo data A. Asymptomaticbacteriuria in C57BL/6 WT mice. Mice were inoculated with 2×10⁵ CFU/mLof ABU 83972, SN25, delta-nlpD or delta-rpoS. Mice were sacrificed after24 hours and bladder tissues were stained with a-Pol IIp Ab. B. Loss PolII phosphorylation is apparent after infection with ABU, this is rescuedafter infection with SN25, d nlpD and d rpoS mutants. This confirms invitro data on NlpD and RpoS being involved in inhibition of Pol IIphosphorylation. C. Neutrophil counts of urine of mice infected withABU, SN25, d nlpD and d rpoS mutants. Functional relevance of Pol IIde-repression in SN25 infected mutant is further suggested by higherneutrophil counts.

EXAMPLE 1 Inhibition of Eukaryotic RNA Pol II Phosphorylation by 83972ABU Strain

The productive mRNA elongation step is generally marked by thephosphorylation of Pol II carboxy terminal domain on Serine-2 residues,consequently, Ser2 phosphorylation of Pol II is a good indicator of itsactivation. As a starting point, flow cytometry was developed as atechnology to quantify Pol II phosphorylation (FIG. 1). Human kidneyepithelial A498 cells were infected with ABU 83972 strain for 4 hoursand after fixing and permeabilization, cells were stained withantibodies against phosphorylated Ser-2 in Pol II and goat anti-rabbitsecondary antibodies labelled with Alexa-fluor 488. A marked reductionin Ser 2 phosphorylation was detected, compared to uninfected cells(FIG. 1A).

This effect was also confirmed using confocal microscopy (FIG. 1B).Infection with E. coli 83972 induced a change in the magnitude anddistribution of phosphorylation, compared to uninfected cells. The lossof Pol II staining was visible as a loss in total fluorescence (meanvalue of 158, 493 AU compared to 588,307 AU, p<0.001), (FIG. 1C). Inaddition, a change in distribution resulted in the emergence of twopeaks, with intensities of 5,522 and 256,779 AU. Uninfected cells showedone single peak with higher fluorescence intensity at 605,108 AU. Pol IIinhibition was also clearly visible using confocal microscopy (FIG. 1B).The results confirm the inhibition of eukaryotic RNA Pol IIphosphorylation by E. coli 83972.

To refine the Pol II phosphorylation data for middle-size events, Pol IIphosphorylation was measured for events within gate R2. A higher numberof cells fell into gate R2 for control compared to ABU infected sample(76.2 and 3 5.2%). ABU infection causes formation of smaller cells orbroken cells (nuclei) when compared to uninfected sample as was seenfrom the number of small-size events. When R2-gated events are takeninto consideration, lower-intensity peak of Pol II fluorescence in ABUsample becomes much less prominent. Mean value of Pol II phosphorylationin control and ABU infected sample for the representative experiment was510,668 and 222,972 AU, respectively.

EXAMPLE 2 Identification of an ABU 83972 Variant SN25 with Loss of PolII Inhibitory Activity and its Genome Sequencing

Patients were inoculated with therapeutic doses of ABU 83972. Theprotocol for therapeutic bladder inoculation of patients with E. coli83972 has been described previously (Agace, J Clin Invest, 1993; Wullt,Mol Microbiol, 2000; Sunden, J Urol, 2010). Briefly, after antibiotictreatment to remove prior infection, patients were inoculated with E.coli 83972 through a catheter (30 ml, 10⁵ cfu/ml in saline). Blood andurine samples were obtained before and repeatedly after inoculation.Throughout the colonization period, viable bacterial counts in urinewere determined, monthly urine samples were collected and analyzed forIL-6 and IL-8 as well as neutrophil infiltration. Bacteria from eachurine sample were verified by PCR for presence of a kryptic plasmidunique for strain 83972 and one chromosomal marker (4.7-kb deletion instrain 83972 in the type 1 fimbrial gene cluster). For further analysis,five independent colonies per urine sample were used.

Re-isolates from inoculated patients were then screened for Pol IIinhibitory activity as described in Example 1. One re-isolate,designated SN25, had lost Pol II inhibitory activity (FIG. 1D). A498cells were infected with ABU or SN25 E. coli strain and labelled forphosphorylated Pol II as described above. Mean values of RNA Pol IIphosphorylation in SN25-infected samples compared to uninfected controlcells and obtained with flow cytometry and confocal microscopy, areshown in (FIGS. 1E and 1F, respectively). The ABU strain suppressed PolII phosphorylation by about 73%, while SN25 by only 19%, suggesting thatsome genes in SN25 genome, responsible for suppression of Pol IIphosphorylation, might have been lost or inactivated.

Genome of SN25 was sequenced (FIG. 2A) to identify responsiblemutations, and 36 genomic changes were found compared to E. coli 83972wt; 25 of them were in coding region with 8 resulting in amino acidchange (FIG. 2B). Among the affected genes are those, responsible forL-lactate metabolism (lldD and lldR), regulation of motility andchemotaxis (lrhA), cell wall formation (nlpD) and biofilm formation(rfaH and cysE) (FIG. 2C). RfaH is a transcriptional anti-terminator,required for efficient expression of long chain LPS expression andhemolysin, its loss attenuates virulence of UPEC. CysE is a serineacetyltransferase, catalyzes the conversion of L-serine toO-acetyl-L-serine. Inactivation of mdoH leads to increased expression ofcolanic acid capsular polysaccharide. RcsB is a positive responseregulator for colanic capsule biosynthesis.

EXAMPLE 3 Screening of Single-Gene Mutants to Identify Genes Responsiblefor Pol II Inhibition

To identify genetic determinants of Pol II phosphorylation, genescomprising the identified variant sequences were replaced in E. coli83972 chromosome by homologous recombination with chloramphenicolresistance cassette. Deletions were validated (Uli). The mutants weresubsequently screened for effects on Pol II (FIG. 3A). Single deletionsof ΔlldD, ΔlldR, ΔnlpD, ΔrfaH and ΔcysE reduced the inhibitory effect ofE. coli 83972 wt, as shown by flow cytometry and confocal microscopy(FIG. 3B-C). Statistical difference in level of Pol II phosphorylationwas measured with t test.

The lldD gene is responsible for aerobic L-lactate metabolism, whoseproduct catalyzes the interconversion of L-lactate and pyruvate, whilelldR is a regulator of the lldPRD operon. It was concluded from thesedata that products of both lldD and lldR genes are responsible forsuppression of RNA Polymerase II phosphorylation. The low-intensity PolII phosphorylation peak is less prominent after infection with SN25, ABUΔlldD and ABU ΔlldR, which is in agreement with higher mean values ofPol II phosphorylation for cells infected with SN25 and ABU mutantscompared to ABU. As shown, mean Pol II phosphorylation for ungatedevents for control cells and cells infected with ABU, SN25, ABU ΔlldDand ABU ΔlldR is 100, 78, 79, 50 and 50%, respectively. Thus, the thatproducts of lldD (p<0.4) and lldR genes lead to inhibition of Pol IIphosphorylation, with effect of lldR being statistically significant(p<0.05).

Several other mutants, which had significant effect on de-repression ofPol II phosphorylation, are 83972ΔnlpD (p<0.01), 83972ΔrfaH (p<0.01) and83972ΔcysE (p<0.05) (FIGS. 3B and C). Gene nlpD encodes a lipoproteinwith a potential function in cell wall formation. Gene rfaH encodes fora transcriptional anti-terminator, required for efficient expression oflong chain LPS, hemolysin and affects biofilm formation. Gene cysEencodes for serine acetyltransferase, which catalyzes the conversion ofL-serine to O-acetyl-L-serine that is the first step of L-cysteinebiosynthesis from L-serine; cysE product also affects biofilm formationin E. coli K-12.

EXAMPLE 4 Secretion of Bacterial Inhibitors of Pol II Phosphorylation

In parallel with the genetic studies, a biochemical approach was takento identify the compound responsible for the Pol II inhibitory activity.Bacteria were incubated for 4 hours in tissue culture medium (RPMIsupplemented with 1 mM pyruvate). The medium was harvested after 4hours, centrifuged at 4,000×g for 10 min and sterile filtered to removeremaining bacteria (0.2 μm filter), before addition to human kidneycells (FIG. 3D). E. coli are typically 2 μm long and 0.5 μm in diameter,and filtration through 0.2 μm syringe filters removes bacterial cells.Significant Pol II inhibitory activity (p=0.023) was identified in theABU culture supernatants compared to uninfected, substituted RPMI,suggesting that growth in cell-free culture medium induced the secretionof the inhibitor(s).

As it was shown that supernatant of ABU bacteria have similar inhibitoryeffect on Pol II as ABU bacteria per se, we questioned if this effect islost in the SN25 supernatant. Phosphorylation of Pol II wassignificantly higher (p<0.05) after incubation with SN25 supernatantcompared to ABU supernatant, suggesting that the strain has lost theability secrete inhibitors. Mutants of SN25 were therefore were grown inRPMI and their supernatants were harvested. Supernatants of lldD, lldR,nlpD and rfaH mutants had lost Pol II inhibitory activity (p<0.02 andp<0.05, respectively) compared to ABU supernatant, (FIG. 3E) indicatingthat genes lldD, lldR and nldD achieve their effect via effectormolecules that are secreted by the bacteria.

The inhibitory activity of the supernatant was shown to be heatsensitive (100° C., 30 min) but Proteinase K resistant. The molecularsize of the inhibitory component was found to be <3 kDa, by centrifugalultrafiltration with a 3 kDa filter. Elevated levels of acetic acid andformic acid were detected in the filtrate of the ABU supernatant, usingion exchange chromatography. A rapid increase in formic-, succinic- andacetic acids was also detected by Mass spectrometry analysis ofmetabolites secreted by ABU upon growth in urine. Finally, highconcentrations of formic and acetic acids were shown to inhibit Pol IIphosphorylation (χ² test for independence compared to medium control).

EXAMPLE 5 The ABU Strain Inhibits the Pol II Ser2 CTD PhosphorylatingMachinery by Targeting Cyclin Kinase 12 and its Recruiting Protein PAF1C

The Pol II phosphorylation complex required to phosphorylate Ser2 isassembled in several steps (FIG. 4A). The preinitiation complexcontaining the TATA box binding protein (TBP), binds to DNA upstream ofthe transcription start site and the activated complex then recruitstranscription factor IID, and the N-terminal Zink ribbon domain of TFIIb is required to recruit Pol II. following the binding of TATA boxbinding to DNA, TBP recruits Pol II and the DNA-binding. The DNA bindingsubunit of Pol II anchors the preformed complex to DNA recruits Pol IIto different eukaryotic promoters and the beta subunit is phosphorylatedby the C-terminal domain (CTD) phosphorylation machinery.Cyclin-dependent kinase 9 (CDK9) brings the adaptor PAF1C into closeproximity with Pol II and the PAF1C subunit CDC73 recruits CDK12 to thecomplex. CDK9 and CDK12 then phosphorylate the Pol II CTD domain, at Ser2 (FIG. 4A).

To examine how the ABU strain suppresses phosphorylation of host RNApolymerase II CTD at Ser2 residue (FIG. 5E), we further analyzedpotential protein targets in the Pol II phosphorylation pathway. Twocyclin dependent kinases are responsible for Pol II phosphorylation,−CDK9 and CDK12 (FIG. 4a ). CDK9 is involved as well, bringing PAF1C inclose proximity to the Pol II promoter complex. PAF1C acts as arecruitment protein for CDK12.

To address how E. coli 83972 inhibits the Pol II phosphorylationmachinery, CDK12, CDC73 (PAF1C subunit) and CDK9 in host cells, afterinfection with ABU and SN25 was quantified by Western Blot analysis(FIG. 4B). CDK12 and PAF1 decreased drastically after ABU infection butremained at control levels after SN25 infection. CDK9 levels were lessstrongly affected (40%). This effect on PAF1C and CDK12 was confirmed byconfocal microscopy (FIG. 4C-D). SN25, in contrast, did notsignificantly alter PAF1C, CDK12 or CDK9 protein levels compared touninfected cells.

To address if the inhibitory activity was a secreted bacterial product,A498 cells were treated with supernatants of cells infected with thesingle gene mutants of the ABU strain, and CDK12 and CDC73 levels weresubsequently tested in WB. The results are shown in FIG. 4E. The nlpDdeletion mutant had lost the inhibitory activity against PAF1 and CDK12.

This was confirmed by confocal microscopy (FIG. 4F). NlpD gene islocated in one gene cluster with rpoS, encoding one of sigma subunits ofbacterial RNA polymerase (FIG. 5A). Remarkably, the list of genesrelated to CDK12 and CDC73 suppression showed a direct association withthe list of genes with effect on Pol II phosphorylation. In contrast,nlpD, lldD, lldR and rfaH mutations did not affect CDK12 or CDC73 andthe cysE, lrhA, mdoH and rcsB deletion mutants resembled SN25.

Overall, these results indicate that nlpD and rpoS suppress Pol IIphosphorylation by targeting the CDK12 and PAF arm of hostphosphorylating machinery.

EXAMPLE 6 Investigation into NlpD and RpoS

In E. coli 83972, NlpD is located upstream of rpoS, which encodes SigmaS; the DNA binding subunit of bacterial RNA Polymerase (FIG. 5A). NlpDregulates Sigma S expression and may facilitate its release byactivating cell wall hydrolases. To address if the effects of NlpD onPol II and PAF are executed through rpoS, we constructed an rpoSdeletion mutant in E. coli 83972. The deletion was confirmed by DNAsequencing and the loss of Sigma S protein was verified by Western blotanalysis of bacterial cell extracts (FIG. 5B). Sigma S was present inextracts from E. coli 83972WT bacteria but was absent in extracts fromSN25, ΔnlpD and ΔrpoS. In bacterial supernatants, the loss of Sigma S inthese strains was confirmed (FIG. 5B).

The applicants subsequently examined the effect of the rpoS mutant onPol II phosphorylation (FIG. 5C). The rpoS mutant had lost the abilityto inhibit Pol II phosphorylation, and Pol II staining in infected cellswas comparable to SN25 and the delta NlpD mutant (n.s.). A similareffect was observed for PAF1C, CDK12 as the rpoS mutant had lost theability to inhibit PAF1C and CDK12 (FIGS. 6C-D). The results suggestthat Sigma S acts as bacterial effector molecules in human cells,responsible for the inhibition of Pol II phosphorylation through PAF1C.The results suggest that the effects of NlpD on RpoS account for theattenuation of Pol II—Ser 2 phosphorylation.

EXAMPLE 7 Subcellular Distribution of nlpD and rpoS in Infected HumanCells

To address if these molecular interactions may be relevant in infectedcells, the applicants subsequently examined, if Sigma S is internalizedinto human cells, after infection with E. coli 83972WT. By Western blotanalysis of whole cell extracts, a single band of 40 kDa was detected,after staining with Sigma S specific antibodies. A band with similarmobility was detected in nuclear extracts, suggesting nucleartranslocation of Sigma S (FIG. 6B). The Sigma S band was not detected incells infected with SN25 or the AnlpD- or ΔrpoS deletion mutants.

The internalization and nuclear translocation of Sigma S was confirmed,by co-immunoprecipitation, using Pol II specific antibodies. A bandcorresponding to Sigma S was detected in extracts from cells infectedwith ABU but not SN25. In a further Pol II co-ip involving whole cellextracts from cells infected with the NlpD or RpoS mutants, Sigma S andNLPD bands were detected in ABU infected cells but not in the singlegene mutants. Low Sigma S in SN25, which does not carry a deletion.

In addition, human kidney cells were infected with E. coli 83972WT andstained with antibodies specific for Sigma S or Pol II, with nuclearDRAQ5 counterstaining. A parallel loss of nuclear Pol II staining andaccumulation of RpoS in nuclear aggregates was detected. In contrast,Sigma S was not detected in cells infected with SN25, ΔrpoS or AnlpD(FIG. 6D).

EXAMPLE 8 Competitive Inhibition of TBP Binding by Sigma S

The Pol II phosphorylation complex required to phosphorylate Ser2 isassembled in several steps (FIG. 4A). The preinitiation complexcontaining the TATA box binding protein (TBP), binds to DNA upstream ofthe transcription start site and the activated complex then recruitstranscription factor IID, and the N-terminal Zink ribbon domain of TFIIb is required to recruit Pol II. following the binding of TATA boxbinding to DNA, TBP recruits Pol II.

Like the TATA-box binding protein in eukaryotes, Sigma S binds to DNAand is the TATA box binding protein of the bacterial RNA Pol II complex(FIG. 5E). We therefore tested the hypothesis that Sigma S may bind toeukaryotic promoter DNA and competitively inhibit TBP binding (FIG. 5F).Human and bacterial TATA box oligonucleotides were incubated with asynthetic peptide comprising the Sigma S DNA-binding domain (aa149-183-SEQ ID NO 2) in an electro-mobility shift assay (EMSA). Sigma Swas shown to bind prokaryotic and human TATA box oligonucleotides,creating band shifts with similar mobility (FIG. 5G). Specificity wasconfirmed by inhibition of the band shift with Sigma S-specificantibodies. The Sigma S peptide was subsequently shown to competitivelyinhibit TBP binding to the IRF3 promoter, in a concentration-dependentmanner (FIG. 5H).

To examine if E. coli 83972 affects PIC formation in infected cells, wequantified the TATA box binding protein (TBP) in total cell extractsfrom uninfected and infected kidney cells (FIG. 5B-D). E. coli 83972reduced the TBP and TF2b protein levels. By confocal microscopy, nucleartranscription factor II b (TF2b) staining was reduced. SN25, incontrast, did not affect the PIC components (FIGS. 5C and D) and thenlpD deletion mutant reproduced the SN25 phenotype (FIG. 5F).

EXAMPLE 9 In Vivo Relevance

The effects on Pol II phosphorylation were confirmed in vivo, in themurine urinary tract infection model. C57BL/6 WT mice were inoculatedwith 2×10⁵ CFU/mL of ABU 83972, SN25, delta-nlpD or delta-rpoS andsacrificed after 24 hours. Tissue levels of RNA Pol II were quantifiedby staining of frozen tissue sections after staining with specificantibodies (FIG. 7A). E. coli 83972 inhibited mucosal Pol II staining inthe urinary bladder mucosa, confirming the cellular studies (p<0.05,FIG. 7B). This inhibitory effect was not detected in mice infected withthe SN25 mutant strain or the delta NlpD or Sigma S mutant strains.Neutrophil counts in urine of infected mice were measured (FIG. 7C) andhigher counts were found in SN25 infected mice, indicating functionalrelevance of Pol II de-repression. Furthermore, it was found that theSN25 infection led to higher level of bacteria in bladder, suggestinghigher virulence.

Materials and Methods Bacterial Strains

Asymptomatic bacteriuria (ABU) strain was isolated during a study ofchildhood UTI in Goteborg, Sweden (Lindberg et al., 1978). Bacteria werecultured on tryptic soy agar (TSA, 16 h, 37° C.) and harvested inphosphate-buffered saline (PBS, pH 7.2). For the course of infection,bacteria were diluted to reach final concentration in medium 1×10⁸cfu/ml.

Cell Culture

Human kidney carcinoma (A498, ATCC HTB44) were cultured in RPMI-1640(Thermo Scientific) supplemented with 1 mM sodium pyruvate, 1 mMnon-essential amino acids (GE Healthcare) and 10% heat-inactivated FBSat 37° C. with 5% CO₂.

Preparation of Bacterial Supernatant

Bacteria were incubated for 4 hours in tissue culture medium, the mediumwas harvested, centrifuged at 4,000×g for 10 min and filtered to removeremaining bacteria. Human kidney cells A498 were incubated with filteredsupernatant for 4 hours.

Cell Flow Cytometry

Before infection, A498 cells were washed twice with RPMI medium withoutFCS. Cells were detached with Versene for 15 min, centrifuged at 400 gfor 5 min and re-suspended in ice-cold PBS. 5×10⁵ cells were treated insuspension as follows: cells were infected, fixed (3.7% formaldehyde inPBS, 15 min), permeabilized (0.25% Triton X-100, 5% FBS in PBS, 10 min),blocked (5% FBS, 1 h at RT), incubated with primary antibodies in 5% FBSovernight at 4° C. (anti-RNA Polymerase II subunit B1 (phospho CTDSer-2) 1:800, Merck) and with fluorescently labeled secondary antibody(Alexa Fluor® 488 goat anti-rat IgG, A-11006, 1:600, Thermo Scientific)for 1 h at RT. After each step above apart from permeabilization andblocking, cells were washed twice with ice-cold PBS and centrifuged at400 g for 5 min. After final wash, cells were re-suspended in flowcytometry buffer 0.02% EDTA 5% FCS in PBS. With BD Accuri C6 flowcytometer (BD Biosciences), 20,000 events were collected at 60 ul/minflow rate.

Confocal Microscopy

Cells were grown to 70-80% confluence on 8-well chamber Permanox® slides(3×10⁴ cells/well, Thermo Fisher Scientific), and infected, fixed,permeabilized and treated with AB as for flow cytometry. After nuclearstaining (15 min, DRAQS, Abcam), slides were mounted (Fluoromount,Sigma-Aldrich), imaged by laser-scanning microscopy (LSM800, Carl Zeiss)and quantified by ImageJ software 1.46r (NIH).

Ion Exchange Chromatography

Organic acids were analyzed on a Dionex anion chromatography system bythe Swedish Environmental Research Institute. Potassium hydroxide wasused as an eluent to separate ions in the sample. To obtain the bestpossible separation the concentration of the eluent was graduallychanged during the process. After separation, a cation exchanger wasused to reduce the conductivity of the eluent and to convert the anionsinto their respective acids.

Global Gene Expression

Total RNA was extracted from A498 cells in RLT buffer with 1%β-Mercaptoethanol. 100 ng of RNA was amplified using GeneChip 3′IVTExpress Kit, 6 ng of fragmented and labeled aRNA was hybridized ontoHuman Genome array strips for 16 hours at 45° C., washed, stained andscanned using the GeneAtlas system (Affymetrix). All samples passed theinternal quality controls included in the array strips (signal intensityby signal to noise ratio; hybridization and labeling controls; samplequality by GAPDH signal and 3′-5′ ratio<3).

Fold change was calculated by comparing cells treated with ABU ormutants to uninfected cells (PBS control) of the same geneticbackground. Significantly altered genes were sorted by relativeexpression (2-way ANOVA model using Method of Moments, P-values <0.05and absolute fold change >1.41) (Eisenhart 1947). Heat-maps wereconstructed with Excel. Differentially expressed genes and regulatedpathways were analyzed by Ingenuity Pathway Analysis software (IPA,Ingenuity Systems, Qiagen) and String and David open source software.

Western Blotting

Cells were lysed with RIPA lysis buffer, supplemented with protease andphosphatase inhibitors (both from Roche Diagnostics). Proteins were runon SDS-PAGE (3-8% or 4-12% Bis-Tris gels, Invitrogen), blotted onto PVDFmembranes (GE Healthcare) blocked with 5% non-fat dry milk (NFDM),incubated with primary antibody: mouse anti-CDK12 (1:400 in 5% NFDM,ab9722, Abcam), mouse anti-Parafibromin (1:400 in 5% BSA, sc-22514-R,Santa Cruz), washed with PBS tween 0.1% and incubated with secondaryantibodies in 5% NFDM (goat anti-mouse-HRP, Cell Signaling). Bands wereimaged using ECL Plus detection reagent (GE Health Care) and quantifiedusing ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loadingcontrol.

1. A pharmaceutical composition comprising an inhibitor of RNApolymerase II and a pharmaceutically acceptable carrier, wherein saidinhibitor targets a protein selected from cyclin kinase 12 (CDK12) orits recruiting protein PAF1C.
 2. The pharmaceutical compositionaccording to claim 1 wherein the inhibitor is a polypeptide expressed bya gene selected from lldD, lldR, nlpD or rfaH of a bacterial species, ora variant of said protein.
 3. The pharmaceutical composition accordingto claim 2 wherein the bacteria is a commensal bacteria or anasymptomatic carrier.
 4. The pharmaceutical composition according toclaim 3 wherein the bacteria is asymptomatic bacteriuria (ABU).
 5. Thepharmaceutical composition according to claim 2 wherein the bacteria isan E. coli strain.
 6. The pharmaceutical composition according to claim5 wherein the bacteria are E. coli
 83972. 7. The pharmaceuticalcomposition according to claim 1 wherein the inhibitor is a Sigma Sprotein or a variant thereof or an active fragment of either of these.8. The pharmaceutical composition according to claim 7 wherein the SigmaS protein is e# SEQ ID NO 1 or SEQ ID NO 2 or SEQ ID NO
 3. 9. Thepharmaceutical composition according to claim 1 wherein the inhibitor isa bacterial NplD protein, or a variant thereof, or an active fragment ofeither of these.
 10. The pharmaceutical composition according to claim 9wherein the bacterial NplD protein is SEQ ID NO
 4. 11. Thepharmaceutical composition according to claim 2 wherein the inhibitor issecreted by the bacteria.
 12. The pharmaceutical composition accordingto claim 9 wherein the inhibitor is of less than 3 kDa in molecularweight.
 13. The pharmaceutical composition according to claim 2 whereinthe inhibitor is synthetic.
 14. (canceled)
 15. A method for preparing aninhibitor of RNA polymerase II that is expressed by a bacterial specieswhich comprises culturing suitable bacteria in a culture medium, andisolating a factor having RNA polymerase II inhibitor activity from thesupernatant.
 16. (canceled)
 17. A method for providingimmunosuppression, anti-inflammatory or anti-infection therapy to apatient in need therefore, the method comprising administering to thepatient an effective amount of an inhibitor of RNA polymerase II. 18-27.(canceled)
 28. The method of claim 17, wherein the inhibitor is apolypeptide expressed by a gene selected from lldD, lldR, nlpD or rfaHof a bacterial species, or a variant of said protein.
 29. The method ofclaim 17, wherein the inhibitor is a bacterial NplD protein, or variantthereof, or active fragment of either of these.
 30. The method of claim29, wherein the bacterial NplD protein is SEQ ID NO
 4. 31. The method ofclaim 17, wherein the inhibitor is a Sigma S protein, or a variantthereof, or an active fragment of either of these.
 32. The method ofclaim 31, wherein the Sigma S protein is SEQ ID NO 1 or SEQ ID NO 2 orSEQ ID NO 3.